Enzymes of hydrogen metabolism in Pyrococcus furiosus

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN o peron for a 14-subunit transmembrane complex associated with a Ni-Fe hydrog enase. Ten ORFs (mbhA-I and mbhM) encode hydrophobic, membrane-spanning sub units. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni-Fe hydroge nase, however, the small subunit can coordinate only a single iron-sulfur c luster, corresponding to the proximal [4Fe-4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe-4S] protein. With P. f uriosus soluble [4Fe-4S] ferredoxin as the electron donor the membranes pro duce H-2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely h igh ratio of H-2 evolution to H-2 uptake activity compared with other hydro genases. The activity is sensitive to inhibition by dicyclohexylcarbodiimid e, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with E-m app roximate to -0.33 V. The genome contains a second putative operon, mbxABCDF GHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni-Fe-cluster in the large hydrogenase subunit. Kinetic studies of memb rane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activ ities allow the formulation of a comprehensive working hypothesis of H-2 me tabolism in P. furiosus in terms of three pools of reducing equivalents (fe rredoxin, NADPH, H-2) connected by devices for transduction, transfer, reco very and safety-valving of energy.